1 Field of the Invention
The present invention relates to the area of viral diagnosis, in particular Epstein-Barr virus (EBV) diagnosis. Improved methods for the detection of EBV infections and agents suitable for this purpose are provided.
2 Brief Description of the Related Art
The Epstein-Barr virus is a human herpes virus which is considered to be the trigger of infectious mononucleosis. Infection with EBV often takes place subclinically, occasionally with mild symptoms. In immunosuppressed persons, however, an EBV infection can lead to severe malignant manifestations.
Importance of Epstein-Barr Virus (EBV) Diagnosis
EBV diagnosis is of considerable differential diagnostic importance since the symptoms of acute EBV infection may overlap with many clinical pictures which are caused by other pathogens or have other causes. Thus, an acute EBV infection may be confused with a primary HIV infection, rubella virus infection, cytomegalovirus infection, infection by classical hepatitis viruses, toxoplasmosis, leptospirosis, infection by various neurotropic pathogens and with leukaemia or a lymphoma. Unambiguous EBV serology therefore constitutes a major obligation to the patient.
Problems of EBV Serology
Classical EBV serology is based on the detection of the IgG and IgM antibody response to viral capsid antigens (VCA) and to EBV-specific nuclear antigens (EBNA), in particular EBNA-1.
The classification of various EBV antigen classes is based on the biological cycle of virus multiplication with proteins required early (EA) and late (VCA) and antigens for maintaining the latency (EBNA). These antigen classes were detectable and distinguishable in the first serological test systems (immunofluorescence with EBV-infected cells). All further subsequent developments of serological test systems retained this scheme and thus permit the distinction of antibody specificities with respect to EBNA, VCA and EA.
The VCA and EBNA markers were first defined in the immunofluorescence technique. In more recent tests, the purified components p18 and p23 of the VCA complex, which are generally prepared by a recombinant method and p72, which corresponds to EBNA-1, are used. The EBNA-1 antigen is formed at a late stage after an initial infection with the establishment of cells with latent infection and is one of the factors responsible for maintaining this status. This means that anti-EBNA-1 is formed only after the elapse of up to six months following initial infection. At the same time, however, it also means that, when this marker is present, diagnosis can safely exclude a recent infection, and thus an important marker for assignment of the time of infection is available.
EBV serology has a high degree of variability, frequently leading to ambiguous or false results if only the classical markers VCA-IgG, VCA-IgM and anti-EBNA-1 are determined. Thus, for example, not all acute EBV infections have a detectable VCA-IgM response, so that in this case test strategies which are based only on the determination of VCA-IgG and VCA-IgM lead to a false conclusion. On the other hand, a VCA-IgM response can in rare cases also persist for months and thus simulate a recent infection.
Positive anti-EBNA-1 is evidence of an elapsed EBV infection and, in combination with positive VCA-IgG, is the only unambiguous serological constellation. Negative anti-EBNA-1 (with simultaneously positive VCA-IgG) either may indicate an acute EBV infection or may be caused by secondary anti-EBNA-1 loss in the case of immunosuppression In addition, about 5% of those infected never develop detectable anti-EBNA-1 and thus simulate the serological situation of an acute EBV infection for their whole life.
Thus, an acute EBV infection is safely ruled out by simultaneous positive VCA-IgG and anti-EBNA-1. However, positive VCA-IgG with negative anti-EBNA-1 represents an ambiguous constellation which occurs in three situations:                1) in case of recent EBV infection;        2) in case of elapsed infection and anti-EBNA-1 loss through suppression of the cellular immune system;        3) in case of elapsed infection and simultaneous lack of anti-EBNA-1 formation (in about 5% of the healthy population).        
Cases 2) and 3) are, for example, to be found more frequently than true recent EBV infection among patients examined in major hospitals.
This key problem of EBV serology could be reliably solved to date only by complicated, additional tests or by repetitions over a period of time in association with clinical data.
The lack of anti-EBNA-1 is therefore generally used as an indicator of a fresh infection. As mentioned above, this can lead to drastic misinterpretations in the case of EBV-infected persons who remain anti-EBNA-1-negative or, because of suppression of the cellular immune system, lose the relevant antibodies. The current methods used in routine diagnosis and employing anti-EBNA-1 do not safely permit the diagnosis of a fresh EBV infection.
The differentiation of primary anti-EBNA-1 negativity in acute EBV infection on the one hand and anti-EBNA-1 loss and lack of anti-EBNA-1 formation on the other hand is therefore the major problem of EBV serology. This particular problem is not taken into account in most published evaluations of serological tests. It must be assumed that up to 20 percent of the examinations for EBV infection are ambiguous and lead to incorrect findings. These are of major importance particularly in oncology, transplantation medicine and care during pregnancy.